1.Write a summary of one of the articles you are reading for your project paragraph by paragraph. One or two sentences per paragraph is fine. You must do this in your own words. No significant amount of text can be copied from the abstract or any part of the paper. Either put the summary in bullet form on your research log or onAcaWiki.Due October 29, 2009 20:50 PM
Article:
Kalanxhi, E., and Wallace, C. J. A. (2007) Cytochrome c impaled: investigation of the extended lipid anchorage of a soluble protein to mitochondrial membrane models, Biochem J 407, 179-187. DOI: 10.1042/BJ20070459
[Full Marks JCB]
Article Summary:
Abstract: •Past investigations have envisioned cytochrome c more so as a surface protein. Within the last few years it was determined that this might not be the case as the protein is now considered to be anchored into the phospholipid bilayer, through the insertion of an acyl chain. This article investigates the conformational changes of cytochrome c as well as the model for lipid anchorage.
Introduction: •Cytochrome c has classically be attributed to electron transport, which is utilized in the formation of ATP. More recently it was found that it plays a significant role in apoptosis, programmed cell death. The exact mechanism is still unknown but it has revived a great interest in this model protein. •The primary assumption of the past was that cytochrome c was bound to the inner mitochondrial membrane through electrostatic interactions, the binding was due to positive patches on the proteins surface. Newer research leads to cytochrome c playing a more dynamic role within the mitochondria, with proteins both loosely and firmly anchored to the lipid bilayer. With the latter found to play a more significant role on the apoptotic process.
•The mechanism of which cytochrome c binding occurs is through cardiolipin [CL], which has been the source of much debate. And is currently under investigation through by a variety of groups at as many angles imaginable.
•Binding occurs at physiological conditions (i.e. low ionic strength), however through lysine residues that are exposed on the surface of the proteins tertiary structure there are other binding mechanisms that could occur as well (cationic), this might be found to play a role as more is understood about cytochrome c.
•Many initial studies have reported a structural change, i.e. monitored changes from the proteins native state, upon protein binding. With the use of CL, DOPS, PC, or PL the results appear to not affect the helical content found in the structure of the protein. As mentioned in the last paragraph the charges surface of the protein may play a role in further binding sites, or the cross-linkage to other liposomes, this is not yet fully understood, however this disruption can be monitored through the heme-Met80 linkage. When the heme iron is in a reduced state the protein is also susceptible to protease digestion, i.e. the tertiary structure is "loosened".
•When the heme Fe is in its oxidized state the binding is more considered to take on the characteristics of lipid insertion. The phosphate group guides this process, inducing cyt c structural changes depending on the environment that it is exposed to. These changes are all completely reversible and can be monitored through the Heme-Met80 linkage.
•Past evidence show an interaction between the hydrophobic environment within the protein, with that of the phospholipid acyl chain, which is referred to as "extended lipid anchorage". However the entry point for this acyl chain insertion has yet to be determined, but seem to be assumed to lie somewhere in the non-native conformational changes, States I-V (State III is the native state), depending on the surface environment.
•This study investigated these interactions between sit c and various liposomes (with CL being of particular interest for my project). The different liposomes represent models for the environment within the mitochondria's inner membrane. With the study closely focusing on ionic strength, pH characteristics, liposomal size and character in order to get a better understanding of the character of the proteins anchor. This paper ultimately will present a model of the process between cyt. c and one of the acyl chains of membrane cardiolipin.
Experimental: •The source of the materials, seems mostly from Sigma-Aldrich, in powder form and then modified or reconstituted. •"Wild type" mutation was made, replacing Cys102, which allows for the direction of binding to one of the lysines within the protein.
•The heme containing fragment was isolated through the digestion of the protein. This heme iron was coordinated to Cys14 and Cys17. Through a semisynthetic pathway this residue was replaced, producing Arg91Nle cyt. c.
•Solutions of cyt. c were prepared on phosphate buffer at a pH of 7.0 (native conditions). With the ferri- and ferro- cytochrome c being separated for use in associated studies.
•PE, PC and CL liposomes were produced, with the method provided, at various pore sizes (50-800 nm) to gain information of the interaction at various surface sizes.
•And finally the binding assay methods which was used to deduce the reaction progress. This was done through the monitoring of CL and phospholipid concentration changes. This was then analyzed using a hill plot representing the cooperative binding process.
Results and Discussion:
•The heme iron transition was monitored from low to high spin, at 550nm where the relative change was experimentally found to be the greatest.
•Incubation changes were made to simulate the environment within the mitochondria. Also it was mentioned that the larger the liposome the smaller the spectral change. •The lipid anchorage was considered to be the rate limiting step due to the conformational changes that were induced as well as the actual binding to the phospholipid surface. This was considered this was because it was assumed that this process would need to overcome a significant thermodynamic barrier. Also denoted was the phospholipid bilayer is not distorted as the cyt. c is bound.
•Physical factors and how they affect the interaction of cyt c with the phospholipid membrane. A variety of conditions were used in order to determine the best representation of the mechanism of cyt c insertion. CL was found to have the strongest binding affinity for the models investigated. •As you move away from biological conditions (i.e. higher ionic strength) the binding of cyt c to the liposomes decreases. It was also denoted that there was a greater amount of change spectrally found at these lower ionic conditions. This is consistent with my finding as well in investigating cyt c in solution at various pH and temperature under low ionic strength. •Electrostatics are an important first step in binding, it was found that affinity increased as conditions were acidified. And this electrostatic binding contributed to a strong binding to the membrane which significantly contributes to the extended anchorage in this two step binding model.
•Comparison of binding strength of different species of cyt c to liposomes. For this stage the CL concentration was held constant and an extensive variety of cyt c sources were examined.
•Upon binding there was little variation among mammalian cyt c, however there was a variation when tuna was investigated. Small differences were noted in surface lysine, so I assume this to mean that heterogeneity was seen under the similar conditions. Since this affinity was also determined to be strong I assume that this holds a particular physiological significance. The iron spin state changes were monitored through Magnetic circular dichroism spectroscopy [MCD].
•Next yeast variant interactions with the membrane were monitored. All of the position 73 mutants showed similar liposomal binding affinity. •Speculation to the removal of the charge on the lys72 residue leads to a decrease in the binding affinity. This reasoning is still not fully understood, but it is most likely causing a distortion that causes a variety of complications with the initial complex, thus lowering the binding affinity overall.
•Next was consideration of the arginine mutants. Apparently this replacement, caused rapid autooxidation. What was interesting in this section was the reduction of binding affinity in residue 58, it was though that this was the site where CL binding occurred and an increase in positive charge on the proteins surface should have increased the binding affinity. This provides a significant argument against this residue as the site of CL binding.
•Lys73 is another significant residue in investigating redox partners (it is also found to ligate with the heme under alkaline conditions solely in solution, denoted as state IV). Cyt c when bound to the membrane is considered to be in a partially unfolded state, with secondary structure still maintained. It is believe that this residue plays a role in the binding equilibrium, removal of this residue is believed to destabilize the proteins interior. •Finally the role of Arg91 and the effect that ATP has on the binding. Arg91 plays an important role in the binding of ATP to cyt c. Obviously then the replacement of this residue had a significant effect on the interaction with the liposomes. THis removes an anionic charge on the surface and allowed for a stabilizing element with the CL complex. •This drew the conclusion that ATP competes with the CL for a binding site on the protein, or this might be a inherent property of the artificial membrane made where multiple CL compete for multiple sites on the proteins surface. •When ATP was introduced the rate of oxidation was decreased, and also introduced a destabilizing element to the interior of the protein.
Conclusion: •Conclusions leading to the mechanistic model to CL membrane models. It is believe that the binding surface is between the acyl chain and the 6th coordinating position on the heme.
•Full insertion must lie in a crevice. The CL is bound at a pivotal head group to Lys72.
•Once the anchoring is made a complex formed is stable. The acyl chain, the linkage group, might flip out in an effort to reduce conformational strain.
•Since the cleft contains the redox partners the monitoring of the electron transfer rate while bound to CL would be an interesting study. This could provide additional insight into the the mechanism of the acyl chain insertion. •Is this of physiological relevance? This study has not been completed in ferricytochrome c as of yet. •Extended lipid anchorage might play a significant role in the proper orientation for cytochrome c function. As seem experimentally this might be the reason why only a small amount are found tightly bound under normal conditions. This can lead to a variety of speculation in cytochrome c's role in the apoptotic process. The investigation into cytochrome c continues, (over 65+ years of research and counting since it was characterized).
References (52)
2. Answer one of the FAQ questions or create and answer an FAQ question relating to a topic relating to chemistry publishing. You must provide at least one relevant reference. You must summarize in your own words - copying text from anywhere verbatim is not allowed.Due October 29, 2009 20:50 PM
Article-level metrics are like an impact factor for journal articles. This will indicate a value to the reader on the quality of the article.
As more journals are created as well as information from other sources, blogs etc, you do not have time to read everything, this would help you to streamline the acquisition or measure of the worth of the article.
PLoS provide additional and regularly updated context, or metrics, to the article, which currently includes data on:
online usage: This denotes the amount of times the article has been "accessed". Articles are provided in three different formats - PDF, HTML, and XML
social bookmarks: Uses CiteULike and Connotea to denote how many times users bookmark a site. This can be used to denote how popular an article is.
comments/notes: Users can provide comments or notes (not anonymously), this interaction is counted as well, or a rating can be provided.
blog posts about the article: Many blogs are written about articles, there are blog agrigators that look into this, such asPostgenomic , Nature Blogs , and Bloglines .
ratings of the article: PLoS allows for "star" ratings of articles where readers can rate the information as they read it.
There is a variety of known issues with article level metrics. The first issue would arise from robots (and meta crawlers) skewing the metric. PLoS has removed a list of robots from its online usage data, but the removal of this is exhaustive and would most likely reamin in some of these metrics. Scopus indercounts the number of citations due to duplicates in the database.
Metrics Example: Grigoryan G, Zhou F, Lustig SR, Ceder G, Morgan D, et al. 2006Ultra-Fast Evaluation of Protein Energies Directly from Sequence. PLoS Comput Biol 2(6):e63.doi:10.1371/journal.pcbi.0020063
The Public Library of Science (PLoS) has a nice website dedicated to this, as they are the initial provider of article level metrics. [JBS]
--- I Updated this in class and just realized I forgot to save it to the main FAQ before I left the workshop ---
Extra Problem Search:
Uvaricin - a bis(tertahydrofuranoid) fatty Acid Lactone that was extracted from Uvaria accuminata .
Scifinder Search revealed 21 references:
Yazbak, A.; Sinha, S. C.; Keinan, E. (1998). J. Org. Chem 63: 5863–5868. doi:10.1021/jo980453a
Structure was confirmed by FT-IR, 1H-NMR, 13C-NMR, COSY in this synthesis paper.
This is the SMILES for [+]-Uvaricin
Article was discovered by means of Scifinder Scholar.
Structure was drawn in ChemDraw to get SMILES, INChI, and SLN (Image shown is from primary source).
A ChemSpider search gave 3 structures, however the stereochemistry was not specified specifically in 2 of them and the 3rd did not match the structure in the article found. Attached is the ID of the structure with the patent link, as mentioned stereochemistry is not specified: ChemSpider ID:3679026
[JBS]
3. Find 5 independent sources of 5 properties associated with a molecule of your choice. Provide all references. Due November 12, 2009 Name: Lithium carbonate [Full Marks JCB] CAS: 554-13-2 BRN: 3999191 PubChem ID: 11125 Molecular Formula: Li2CO3 MW: 73.891 g/mol
Main Page | Log Book | Assignment(s) Page | Class Notes
1. Write a summary of one of the articles you are reading for your project paragraph by paragraph. One or two sentences per paragraph is fine. You must do this in your own words. No significant amount of text can be copied from the abstract or any part of the paper. Either put the summary in bullet form on your research log or on AcaWiki. Due October 29, 2009 20:50 PM
Article:
Kalanxhi, E., and Wallace, C. J. A. (2007) Cytochrome c impaled: investigation of the extended lipid anchorage of a soluble protein to mitochondrial membrane models, Biochem J 407, 179-187. DOI: 10.1042/BJ20070459
[Full Marks JCB]Article Summary:
Abstract:
• Past investigations have envisioned cytochrome c more so as a surface protein. Within the last few years it was determined that this might not be the case as the protein is now considered to be anchored into the phospholipid bilayer, through the insertion of an acyl chain. This article investigates the conformational changes of cytochrome c as well as the model for lipid anchorage.
Introduction:
• Cytochrome c has classically be attributed to electron transport, which is utilized in the formation of ATP. More recently it was found that it plays a significant role in apoptosis, programmed cell death. The exact mechanism is still unknown but it has revived a great interest in this model protein.
• The primary assumption of the past was that cytochrome c was bound to the inner mitochondrial membrane through electrostatic interactions, the binding was due to positive patches on the proteins surface. Newer research leads to cytochrome c playing a more dynamic role within the mitochondria, with proteins both loosely and firmly anchored to the lipid bilayer. With the latter found to play a more significant role on the apoptotic process.
• The mechanism of which cytochrome c binding occurs is through cardiolipin [CL], which has been the source of much debate. And is currently under investigation through by a variety of groups at as many angles imaginable.
• Binding occurs at physiological conditions (i.e. low ionic strength), however through lysine residues that are exposed on the surface of the proteins tertiary structure there are other binding mechanisms that could occur as well (cationic), this might be found to play a role as more is understood about cytochrome c.
• Many initial studies have reported a structural change, i.e. monitored changes from the proteins native state, upon protein binding. With the use of CL, DOPS, PC, or PL the results appear to not affect the helical content found in the structure of the protein. As mentioned in the last paragraph the charges surface of the protein may play a role in further binding sites, or the cross-linkage to other liposomes, this is not yet fully understood, however this disruption can be monitored through the heme-Met80 linkage. When the heme iron is in a reduced state the protein is also susceptible to protease digestion, i.e. the tertiary structure is "loosened".
• When the heme Fe is in its oxidized state the binding is more considered to take on the characteristics of lipid insertion. The phosphate group guides this process, inducing cyt c structural changes depending on the environment that it is exposed to. These changes are all completely reversible and can be monitored through the Heme-Met80 linkage.
• Past evidence show an interaction between the hydrophobic environment within the protein, with that of the phospholipid acyl chain, which is referred to as "extended lipid anchorage". However the entry point for this acyl chain insertion has yet to be determined, but seem to be assumed to lie somewhere in the non-native conformational changes, States I-V (State III is the native state), depending on the surface environment.
• This study investigated these interactions between sit c and various liposomes (with CL being of particular interest for my project). The different liposomes represent models for the environment within the mitochondria's inner membrane. With the study closely focusing on ionic strength, pH characteristics, liposomal size and character in order to get a better understanding of the character of the proteins anchor. This paper ultimately will present a model of the process between cyt. c and one of the acyl chains of membrane cardiolipin.
Experimental:
• The source of the materials, seems mostly from Sigma-Aldrich, in powder form and then modified or reconstituted.
• "Wild type" mutation was made, replacing Cys102, which allows for the direction of binding to one of the lysines within the protein.
• The heme containing fragment was isolated through the digestion of the protein. This heme iron was coordinated to Cys14 and Cys17. Through a semisynthetic pathway this residue was replaced, producing Arg91Nle cyt. c.
• Solutions of cyt. c were prepared on phosphate buffer at a pH of 7.0 (native conditions). With the ferri- and ferro- cytochrome c being separated for use in associated studies.
• PE, PC and CL liposomes were produced, with the method provided, at various pore sizes (50-800 nm) to gain information of the interaction at various surface sizes.
• And finally the binding assay methods which was used to deduce the reaction progress. This was done through the monitoring of CL and phospholipid concentration changes. This was then analyzed using a hill plot representing the cooperative binding process.
Results and Discussion:
• The heme iron transition was monitored from low to high spin, at 550nm where the relative change was experimentally found to be the greatest.
• Incubation changes were made to simulate the environment within the mitochondria. Also it was mentioned that the larger the liposome the smaller the spectral change.
• The lipid anchorage was considered to be the rate limiting step due to the conformational changes that were induced as well as the actual binding to the phospholipid surface. This was considered this was because it was assumed that this process would need to overcome a significant thermodynamic barrier. Also denoted was the phospholipid bilayer is not distorted as the cyt. c is bound.
• Physical factors and how they affect the interaction of cyt c with the phospholipid membrane. A variety of conditions were used in order to determine the best representation of the mechanism of cyt c insertion. CL was found to have the strongest binding affinity for the models investigated.
• As you move away from biological conditions (i.e. higher ionic strength) the binding of cyt c to the liposomes decreases. It was also denoted that there was a greater amount of change spectrally found at these lower ionic conditions. This is consistent with my finding as well in investigating cyt c in solution at various pH and temperature under low ionic strength.
• Electrostatics are an important first step in binding, it was found that affinity increased as conditions were acidified. And this electrostatic binding contributed to a strong binding to the membrane which significantly contributes to the extended anchorage in this two step binding model.
• Comparison of binding strength of different species of cyt c to liposomes. For this stage the CL concentration was held constant and an extensive variety of cyt c sources were examined.
• Upon binding there was little variation among mammalian cyt c, however there was a variation when tuna was investigated. Small differences were noted in surface lysine, so I assume this to mean that heterogeneity was seen under the similar conditions. Since this affinity was also determined to be strong I assume that this holds a particular physiological significance. The iron spin state changes were monitored through Magnetic circular dichroism spectroscopy [MCD].
• Next yeast variant interactions with the membrane were monitored. All of the position 73 mutants showed similar liposomal binding affinity.
• Speculation to the removal of the charge on the lys72 residue leads to a decrease in the binding affinity. This reasoning is still not fully understood, but it is most likely causing a distortion that causes a variety of complications with the initial complex, thus lowering the binding affinity overall.
• Next was consideration of the arginine mutants. Apparently this replacement, caused rapid autooxidation. What was interesting in this section was the reduction of binding affinity in residue 58, it was though that this was the site where CL binding occurred and an increase in positive charge on the proteins surface should have increased the binding affinity. This provides a significant argument against this residue as the site of CL binding.
• Lys73 is another significant residue in investigating redox partners (it is also found to ligate with the heme under alkaline conditions solely in solution, denoted as state IV). Cyt c when bound to the membrane is considered to be in a partially unfolded state, with secondary structure still maintained. It is believe that this residue plays a role in the binding equilibrium, removal of this residue is believed to destabilize the proteins interior.
• Finally the role of Arg91 and the effect that ATP has on the binding. Arg91 plays an important role in the binding of ATP to cyt c. Obviously then the replacement of this residue had a significant effect on the interaction with the liposomes. THis removes an anionic charge on the surface and allowed for a stabilizing element with the CL complex.
• This drew the conclusion that ATP competes with the CL for a binding site on the protein, or this might be a inherent property of the artificial membrane made where multiple CL compete for multiple sites on the proteins surface.
• When ATP was introduced the rate of oxidation was decreased, and also introduced a destabilizing element to the interior of the protein.
Conclusion:
• Conclusions leading to the mechanistic model to CL membrane models. It is believe that the binding surface is between the acyl chain and the 6th coordinating position on the heme.
• Full insertion must lie in a crevice. The CL is bound at a pivotal head group to Lys72.
• Once the anchoring is made a complex formed is stable. The acyl chain, the linkage group, might flip out in an effort to reduce conformational strain.
• Since the cleft contains the redox partners the monitoring of the electron transfer rate while bound to CL would be an interesting study. This could provide additional insight into the the mechanism of the acyl chain insertion.
• Is this of physiological relevance? This study has not been completed in ferricytochrome c as of yet.
• Extended lipid anchorage might play a significant role in the proper orientation for cytochrome c function. As seem experimentally this might be the reason why only a small amount are found tightly bound under normal conditions. This can lead to a variety of speculation in cytochrome c's role in the apoptotic process. The investigation into cytochrome c continues, (over 65+ years of research and counting since it was characterized).
References (52)
2. Answer one of the FAQ questions or create and answer an FAQ question relating to a topic relating to chemistry publishing. You must provide at least one relevant reference. You must summarize in your own words - copying text from anywhere verbatim is not allowed. Due October 29, 2009 20:50 PM
Article-level metrics are like an impact factor for journal articles. This will indicate a value to the reader on the quality of the article.
As more journals are created as well as information from other sources, blogs etc, you do not have time to read everything, this would help you to streamline the acquisition or measure of the worth of the article.
PLoS provide additional and regularly updated context, or metrics, to the article, which currently includes data on:
citations: provided by Scopus , PubMed Central and CrossRef are used to represent the number of citations
online usage: This denotes the amount of times the article has been "accessed". Articles are provided in three different formats - PDF, HTML, and XML
social bookmarks: Uses CiteULike and Connotea to denote how many times users bookmark a site. This can be used to denote how popular an article is.
comments/notes: Users can provide comments or notes (not anonymously), this interaction is counted as well, or a rating can be provided.
blog posts about the article: Many blogs are written about articles, there are blog agrigators that look into this, such asPostgenomic , Nature Blogs , and Bloglines .
ratings of the article: PLoS allows for "star" ratings of articles where readers can rate the information as they read it.
There is a variety of known issues with article level metrics. The first issue would arise from robots (and meta crawlers) skewing the metric. PLoS has removed a list of robots from its online usage data, but the removal of this is exhaustive and would most likely reamin in some of these metrics. Scopus indercounts the number of citations due to duplicates in the database.
Metrics Example: Grigoryan G, Zhou F, Lustig SR, Ceder G, Morgan D, et al. 2006 Ultra-Fast Evaluation of Protein Energies Directly from Sequence. PLoS Comput Biol 2(6): e63. doi:10.1371/journal.pcbi.0020063
The Public Library of Science (PLoS) has a nice website dedicated to this, as they are the initial provider of article level metrics. [JBS]
Extra Problem Search:
Uvaricin - a bis(tertahydrofuranoid) fatty Acid Lactone that was extracted from Uvaria accuminata .
Scifinder Search revealed 21 references:- Yazbak, A.; Sinha, S. C.; Keinan, E. (1998). J. Org. Chem 63: 5863–5868. doi:10.1021/jo980453a
Structure was confirmed by FT-IR, 1H-NMR, 13C-NMR, COSY in this synthesis paper.This is the SMILES for [+]-Uvaricin
SMILES:
O[C@@H]([C@@H]1O[C@@H]([C@@H]2O[C@]([C@@H](OC(C)=O)CCCCCCCCCC)([H])CC2)CC1)CCCCCCCCCCCCC3=C[C@H](C)OC3=O
INChI:
InChI=1/C39H68O7/c1-4-5-6-7-8-15-18-21-24-35(44-31(3)40)36-27-28-38(46-36)37-26-25-34(45-37)33(41)23-20-17-14-12-10-9-11-13-16-19-22-32-29-30(2)43-39(32)42/h29-30,33-38,41H,4-28H2,1-3H3/t30-,33+,34+,35-,36+,37+,38+/m0/s1/i1-12,2-12,3-12,4-12,5-12,6-12,7-12,8-12,9-12,10-12,11-12,12-12,13-12,14-12,15-12,16-12,17-12,18-12,19-12,20-12,21-12,22-12,23-12,24-12,25-12,26-12,27-12,28-12,29-12,30-12,31-12,32-12,33-12,34-12,35-12,36-12,37-12,38-12,39-12,40-16,41-16,42-16,43-16,44-16,45-16,46-16
SLN:
OHC[S=N]H(C[9:S=N]HOC[S=N]H(C[15:S=N]HOC[S=I](C[S=N]H(OC(CH3)=O)CH2CH2CH2CH2CH2CH2CH2CH2CH2CH3)(H)CC@15)CC@10)CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2C[4]=CC[S=I]H(CH3)OC(@5)=O
Article was discovered by means of Scifinder Scholar.
Structure was drawn in ChemDraw to get SMILES, INChI, and SLN (Image shown is from primary source).
A ChemSpider search gave 3 structures, however the stereochemistry was not specified specifically in 2 of them and the 3rd did not match the structure in the article found. Attached is the ID of the structure with the patent link, as mentioned stereochemistry is not specified:
ChemSpider ID: 3679026
[JBS]
3. Find 5 independent sources of 5 properties associated with a molecule of your choice. Provide all references. Due November 12, 2009
Name: Lithium carbonate
[Full Marks JCB]
CAS: 554-13-2
BRN: 3999191
PubChem ID: 11125
Molecular Formula: Li2CO3
MW: 73.891 g/mol
(Data in PDF)
(1588 Hits)
(Data in PDF)
(Data in PDF)
(Data in PDF)
(Data in PDF)
(Data in PDF)
[po - rat]